1. Field of the Invention
This invention concerns a novel method for the preparation of large unilamellar liposomal vesicles. The method is fast, simple, economical, reproducible and convenient and avoids lengthy and harsh chemical and physical conditions and procedures which are detrimental to the lipids and/or drugs or substances to be encapsulated therein.
2. Related Disclosures
Liposomes are now recognized as a drug delivery system which can improve the therapeutic activity and safety of a wide range of compounds. A prerequisite to the successful development and commercialization of liposome products is the capability to scale up production methods in compliance with currently accepted good manufacturing practices, at acceptable cost and using processes which provide the high degree of reproducibility required for finished pharmaceuticals and are void of conditions and manipulations which would destroy, modify or otherwise deactivate these pharmaceuticals.
A number of review, for example Liposomes as Drug Carriers, Wiley and Sons, New York (1988), Liposomes from Biophysics to Therapeutics, Marcel Dekker, New York (1987) have described studies of liposome production methodology and properties, their use as carriers for drugs and interaction with a variety of cell types and pointed out the fact that liposome behavior can vary substantially with certain formulation variables, most notably it would depend on chemical composition, size, surface charge, and drug payload. Therefore, it is desirable to have available a method to form liposomes using processes which control these variables.
Liposomes are small closed spherical structures formed from phospholipids (PLs) in aqueous solutions. Liposomes which are formed by single lipid bilayer are called unilamellar vesicles; those formed by multiple bilayers are called multilamellar vesicles (MLVs). Each liposomal layer is formed by a single phospholipid bilayer which separates internal and external solutions and which bilayer is able to entrap the solvent or drug solute in liposome interior or in the space between two liposomal bilayers in case of MLVs. Unilamellar vesicles may be small liposomes (SUVs) or large liposomes (LUVs).
SUVs and LUVs are important in the studies of membranes, membrane proteins, and as delivery vehicles for drugs and genetic material into cells, and many different methods for their preparation exist. However, most of these methods are time consuming, uneconomical, not reproducible, and require relatively demanding laboratory equipment. These methods involve the exposure of PLs and substance to be encapsulated in liposomes to physical stresses such as sonication, or high hydrostatic pressures and/or exposure to a severe chemical environment such as organic solvents, detergents, low/high pH, all of which may harm these sensitive substances.
Thus, a simple, quick, economical, reproducible and harmless method for vesicle preparation is still being searched for and would be convenient.
Large unilamellar vesicles (LUVs) provide a number of important advantages as compared to MLVs including high encapsulation of water soluble drugs, economy of lipid and reproducible drug release rates. However, LUVs are perhaps the most difficult type of liposomes to produce. "Large" in the context of liposomes usually means any structure larger than 0.1 micron; thus large unilamellar vesicles refers to vesicles bounded by a single bilayer membrane that are above 0.1 micron in diameter.
Two primary methods are used to produce LUVs. One involves the detergent dialysis, the other involves the formation of a water-in-oil emulsion. A number of other techniques for producing LUVs have been reported including freeze-thaw cycling, J. Biol. Chem., 252:7384 (1977), slow swelling in nonelectrolytes, J. Cell Physiol. 73:49 (1969), dehydration followed by rehydration, BBA: 816:1 (1985) and dilution or dialysis of lipids in the presence of chaotropic ions Biochemistry, 22:855 (1983). All these methods, however, show various disadvantages and may not be suitable for large scale pharmaceutical preparations.
Removal of detergent molecules from aqueous dispersions of phospholipid/detergent mixed micelles represents one way for producing liposomes, in particular LUVs. As the detergent is removed, the micelles become progressively richer in phospholipid and finally coalesce to form closed, single bilayer vesicles. Shortcomings of this approach include leakage and dilution of the drug during liposome formation, the danger of drug modification or deactivation by detergent, high cost and quality control of the detergent and the difficulty of removing the last traces of the detergent once liposomes have formed. Three major methods of detergent removal appropriate for this purpose of forming liposomes have been described, namely dialysis, column chromatography and by using Bio-Beads.
Detergent dialysis as a method for liposome preparation is described in J. Biol. Chem., 246:5477 (1971). Detergents commonly used for this purpose include the bile salts and octylglucoside. During dialysis, liposomes are formed in the 0.003-0.2 micron diameter range within a few hours. The procedure requires the use of continuous pumping of buffer or several changes of dialysate if the dialysis is performed in the dialysis bags.
The formation of 100 nm single layered phospholipid vesicles LUVs) during removal of detergent deoxycholate by column chromatography has been reported in Biochemistry, 18:145 (1979). The method calls for mixing phospholipid, in the form of either small sonicated vesicles or a dry lipid film, with deoxycholate at a molar ratio of 1:2, respectively. Subsequent removal of the detergent during passage of the dispersion over a Sephadex G-25 column results in the formation of uniform 100 nm vesicles that are readily separable from small sonicated vesicles. Again, this is a lengthy and laborious process of preparing LUVs.
Another method for forming reconstituted membranes reported in J. Eur. Biochem., 75:4194 (1978) may also be applicable to LUVs preparation. The system involves the removal of a nonionic detergent, Triton X-100, from detergent/phospholipid micellar suspensions. This method is based on the ability of Bio-Beads SM-2 to absorb Triton X-100 rapidly and selectively. Following absorption of the detergent, the beads are removed by filtration. The final particle size appears to depend on the conditions used including lipid composition, buffer composition, temperature, and, most critically, the amount and detergent-binding activity of the beads themselves. While this method seems to be fastest of all above, it still includes impractical manipulation such as handling the beads and filtration not to mention possible contamination of sample with impurities which are often introduced by the use of nonrecrystallized detergent.
LUVs can also be prepared by reverse phase evaporation technique (REV), by forming a water-in-oil emulsion of phospholipids and buffer in presence of an excess organic phase followed by removal of the organic phase under reduced pressure. REV method is disclosed in U.S. Pat. No. 4,235,871. The two phases are usually emulsified by sonification or by other mechanical means. Removal of the organic solvent under vacuum causes the phospholipid-coated droplets of water to coalesce and eventually form a viscous gel. Removal of the final traces of solvent under high vacuum or mechanical disruption, such as vortexing, results in the collapse of the gel into a smooth, nonviscous suspension of LUVs. With some lipid compositions, the transition from emulsion to LUV suspension is so rapid that the intermediate gel phase appears not to form. This method has gained widespread use for liposome formulation which require high encapsulation of a water soluble drug. Drug solute entrapment efficiencies up to 65% were reported.
To prepare REV-type liposomes, the phospholipids are first dissolved in an organic solvent such as diethylether, isopropylether, or their mixtures. The aqueous phase containing the material to be entrapped is added directly to the phospholipid-solvent mixture forming a two-phase system. The two phases are sonicated for a few minutes forming the water-in-oil emulsion, and the organic phase is carefully removed under a partial vacuum. The pressure is usually maintained at about 500 mm Hg for the removal of the bulk of the organic phase (using a nitrogen gas bleed to regulate the vacuum) and then lowered cautiously to complete solvent stripping. Removal of the last traces of solvent transforms the gel into LUVs. Such LUVs have been used to encapsulate both small and large chemical molecules. Biologically active macromolecules such as RNA and various enzymes have been encapsulated without loss of activity.
The principal disadvantage of the REV and other methods described supra is the exposure of the material to be encapsulated in liposomes to organic solvents and to mechanical agitation, both of which can lead to denaturation of proteins, introduction of nicks into nucleic acid strands, modification of chemical entities, and to overall change in biological activity.
Moreover, as is apparent from the above description, all other known and available methods for preparation of LUV's are lengthy, laborious and involve the use of strong chemicals such as for example detergents and organic solvents in combination with procedures for their removal, such as dialysis, column chromatography, biobeads, absorption, filtration, freeze drying, etc. The other methods use intrusive procedures such as REV technique, and require the presence of a very strong organic solvents such as for example, diethylether, isopropylether, trichlorotrifluoromethane, etc.
Disadvantages of such procedures and treatments are apparent since the chemically sensitive molecules such as many pharmaceutical drugs, can hardly withstand an exposure to detergents or organic solvents or to treatment using mechanical agitation, sonication, high shear mixing accompanied unavoidably with high temperatures, filtration, dialysis, high pressure, etc. Treatments using the above means can alter the chemical entity of the drug, often irreversibly and thus change its intended therapeutical use.
Thus it would be highly advantageous to have a simple method for preparation of LUVs which would allow avoidance of both strong chemicals and harsh mechanical procedures.
It has been shown previously that in the aqueous solutions, phospholipid molecules form self closed spherical structures where one or several phospholipid bilayers entrap part of the solvent in its/their interior, and that MLVs are formed spontaneously when dry films composed of neutral phospholipid(s) are hydrated and swollen in excess water by gentle shaking J. Mol. Biol., 8:660 (1964). In contrast to the finite swelling behavior of uncharged neutral films, charged phospholipid films exhibit infinite swelling in excess of water, and the spontaneous formation of heterogeneous populations of vesicles. By using similar procedures, fairly homogeneous preparations of SUVs have been reported, BBA, 896:117 (1987), with however, unavoidably large losses of phospholipids. Moreover, in general, SUVs are not very suitable for economical encapsulation of expensive water soluble pharmaceutical drugs because of their small internal volume.
It is, therefore, a primary object of this invention to provide a simple, fast, economical, reproducible and convenient method for preparation of very large liposomes, in the form of large unilamellar vesicles primarily of around 1 micron size.
A very limited scope of this invention was published by inventors in JACS, 110:970 (1988) on Feb. 3, 1988.